Considerable evidence indicates that differences in metabolic processing of chemical toxins are critical in determining both species and organ sensitivity to individual compounds. The microsomal cytochrome P-450 system catalyzes the oxidation of a variety of substrates and consists of a family of isozymes supported by other reductase proteins. Previous studies characterized the cytochrome P-450 system using the carcinogen acetylaminofluorene (AAF) which is oxidized in the 1, 3, 5, 7, 9 position on the fluorene ring and on the nitrogen. In addition, the effects of a variety of inducers and inhibitors on the kinetics of AAF oxidation in rat and rabbit liver microsomes were examined. More recent studies examined AAF metabolism by control and induced rabbit liver microsomes and by 6 highly purified cytochrome P-450 isozymes (forms 1, 3b, 3c, 3v, 4 and 6) from rabbit liver. Only the formation of 7-hydroxy AAF showed biphasic kinetics, indicative of metabolism by multiple forms of cytochrome P-450, whereas formation of 1-, 3-, 5- and N-hydroxy AAF were monophasic. The kinetics of N-hydroxylation was almost identical with control and induced microsomes and with form 4 of cytochrome P-450. All isozymes examined oxidized AAF in the seven position although Km's and Vmax's varied considerably between forms. AAF metabolism was also examined in human liver microsomes and compared with the hydroxylation of debrisoquine, a substrate with documented polymorphic metabolism in humans. No correlation between AAF metabolism, cytochrome P-450 levels or dibrisoquine 4-hydroxylation was seen. However, the rate of formation of N-, 1-, 3-, 7- and 9-hydroxy AAF appeared polymorphic suggesting the presence of two populations of metabolizers among the human subjects examined. The hydroxylation of AAF also did not correlate with bufuralol oxidation or aldrin epoxidation. These studies have further characterized the differential metabolism of AAF by hepatic cytochrome P-450 isozymes and indicate that this substrate may be a useful proble for studying the role of specific isozymes in xenobiotic metabolism.